This proposal focuses on the signal transduction cascades that are used to wire the nervous system. Without question the brain and spinal cord form the most complicated organ, functioning as the biological supercomputer to control everything in the body, from sensing the environment and initiating movement, to learning, memory, speech and behavior. What is most amazing is that this supercomputer self-assembles during development as each neuron sends out a thin wire-like extension, the axon, which can travel great distances to reach its target. The Eph receptors and their membrane-anchored ephrin ligands play important roles in guiding axons to their targets. In addition to axon pathfinding, Ephs and ephrins control many other cell-cell interactions, including those that occur during hindbrain segmentation and cardiovascular development. Eph receptors have a cytoplasmic protein-tyrosine kinase catalytic domain, while the B-subclass ephrins have a short cytoplasmic domain. Our previous genetic and biochemical studies were the first to demonstrate that when Eph receptor-expressing cells contact ephrin-expressing cells, both molecules become tyrosine phosphorylated and both send signals into their respective cell. Over the past five years, our hypothesis that ephrins and Eph receptors transduce bidirectional signals has become a key feature in the study of this large family of 14 receptors and 8 ephrins. In addition to ongoing biological studies of Eph/ephrin functions, we have focused on defining the biochemistry of this bidirectional cell-cell communication system to understand how these signals are transduced at the molecular level. We have identified a number of proteins that physically associate with the cytoplasmic domains of the ephrins and Eph receptors. These molecules contain important protein-protein interaction domains involved in signal transduction and subcellular localization, including Src homology 2 (SH2) domains (which bind phosphotyrosine sequences), SH3 domains (which bind poly-proline sequences) and PDZ domains (which bind the carboxy-terminus of certain proteins). By identifying and characterizing proteins that physically associate with ephrins and Eph receptors, our long-term objective is to define the signal transduction cascades and cellular responses initiated by bidirectional signaling. In addition to increasing our general knowledge about biochemical signal transduction cascades that control cell-cell interactions and axon guidance, these studies may provide insight into potential molecular targets that may be used to develop therapies of the future, such as those needed to regenerate severed connections following a spinal cord injury.